Current treatments utilizing surgery, radiation therapy, and systemic chemotherapy have done little to alter the natural outcome of many malignant tumors of the central nervous system.
The use of cytotoxic products in the treatment of cancer is well known. The difficulties associated with such treatment are also well known. Of these difficulties, the lack of cancer-specific cytotoxicity has received considerable attention, albeit resolution of these difficulties has met with marginal success. Cytotoxic products kill normal cells as well as cancer cells. Such non-specificity results in a number of undesirable side effects for patients undergoing cancer chemotherapy with cytotoxic products, including nausea, vomiting, diarrhea, hemorrhagic gastroenteritis, and hepatic and renal damage. Due to normal cell toxicity, the therapeutic dosage of cytotoxic products has been limited such that cancerous cells are not killed to a sufficient level that subsequently prevents or delays new cancerous growth.
Current approaches to cancer chemotherapy and other immunological therapies focus on the use of cell-specific antibodies bonded to toxins in order to kill specific populations of cancer cells. Immunotoxins (protein toxins chemically linked to tumor-specific monoclonal antibodies or other ligands) offer potential advantages over more conventional forms of treatment by having higher tumor specificity. Ideally, immunotoxins should discriminate to a high degree between target and non-target cells. The critical point, then, is the development of immunotoxins that are highly toxic for specific populations of cells.
Monoclonal antibodies linked to toxic proteins (immunotoxins) can selectively kill some tumor cells in vitro and in vivo. However, reagents that combine the full potency of the native toxins with the high degree of cell-type selectivity of monoclonal antibodies have not previously been designed.
Immunotoxins may be particularly efficacious for the treatment of neoplastic disease confined to compartments such as the peritoneum or intrathecal space. Direct delivery into the compartment avoids complications associated with systemic delivery and produces relatively high local concentrations, thereby achieving greater therapeutic effects. The cerebrospinal fluid compartment may be amenable to this type of compartmentalized immunotoxin treatment. Zovickian and Youle, J Neurosurg, in press, examined the therapeutic effect of a monoclonal antibody-ricin immunotoxin delivered directly into the CSF compartment in a guinea pig model of leptominingeal neplasia. The immunotoxin therapy extended survival, corresponding to a 2-5 log kill of tumor cells, without detectable toxicity.
Protein toxins used in the constructions of immunotoxins have an A and a B subunit. The A subunit catalyzes the inactivation of protein synthesis, resulting ultimately in cell death. The B subunit has two functions: it is responsible for toxin binding to the cell surface, and it facilitates the translocation of the A chain across the membrane and into the cytosol, where the A chain acts to kill cells.
Previously, two general types of immunotoxins have been used. Immunotoxins made with the complete toxin molecule, both A and B chains, have the complication of non-specific killing mediated by the toxin B chain binding site. This can be avoided by eliminating the B chain and linking only the A chain to the antibody. However, A chain immunotoxins, although more specific, are much less toxic to tumor cells. The B chain, in addition to having a binding function, also has an entry function, which facilitates the translocation of the A chain across the membrane and into the cytosol. Since A-chain immunotoxins lack the entry function of the B chain, they are less toxic than their intact toxin counterparts containing the complete B chain. An ideal toxin for immunotoxin construction would contain the A chain enzymatic function and the B chain translocation function, but not the B chain binding function.
Two heretofore inseparable activities on one polypeptide chain of diphtheria toxin and ricin account for the failure to construct optimal reagents. The B-chains facilitate entry of the A-chain to the cytosol, allowing immunotoxins to kill target cells efficiently and bind to receptors present on most cells, imparting immunotoxins with a graft degree of non-target-cell toxicity.
Some toxins have been modified to produce a suitable immunotoxin. The two best known are ricin and diphtheria toxin. Antibodies which bind cell surface antigens have been linked to diphtheria toxin and ricin, forming a new pharmacologic class of cell type-specific toxins. Ricin and diphtheria toxin are 60,000 to 65,000 dalton proteins with two subunits: the A-chain inhibits protein synthesis when in the cytosol, and the B-chain binds cell surface receptors and facilitates passage of the A subunit into the cytosol. Two types of antibody-toxin conjugates (immunotoxins) have been shown to kill antigen-positive cells in vitro. Immunotoxins made by binding only the toxin A subunit to an antibody have little non-target cell toxicity, but are often only minimally toxic to antigen-positive cells. Another type of immunotoxin is made by linking the whole toxin, A and B subunits, to the antibody and blocking the binding of the B subunit to prevent toxicity to non-target cells. For ricin, the non-target cell binding and killing can be blocked by adding lactose to the culture media or by steric restraint imposed by linking ricin to the antibody. Intact ricin immunotoxins may have only 30-to 100- fold selectivity between antigen-positive and negative cells. but they are highly toxic, and the best reagents can specifically kill a great many target cells.
Intact ricin and ricin A-chain immunotoxins have been found to deplete allogenic bone marrow of T cells, which can cause graft-versus-host diseases (GVHD), or to deplete autologous marros of tumor cells.
Diphtheria toxin is composed of two disulfide-linked subunits: the 21,000 dalton A-chain inhibits protein synthesis by catalyzing the ADP-riboxylation of elongation factor 2, and the 37,000-dalton B-chain binds cell surface receptors and facilitate transport of the A-chain to the cytosol. A single molecule of either a diphtheria toxin A-chain or a ricin A-chain in the cytosol is sufficient to kill a cell. The combination of these three activities, binding, translocation, and catalysis, produces the extreme potency of these proteins. The cell surface-binding domain and the phosphate-binding site are located within the carboxyl-terminal 8-kDa cyanogen bromide peptide of the B-chain. Close to the C-terminus region of the B-chain are several hydrophobic domains that can insert into membranes at low pH and appear to be important for diphtheria toxin entry.
Antibodies directed against cell surface antigens have been linked to intact diphtheria toxin or its A subunit to selectively kill antigen-bearing target cells. Antibody-toxin (immunotoxins) or ligand toxin conjugates containing only the diphtheria A-chain have relatively low cytotoxic activity. Intact diphtheria toxin conjugates can be very potent, but can also have greater toxicity to normal cells. Since the B-chain appears to facilitate entry of the A-chain to the cytosol, it is possible that its presence in whole toxin conjugates renders them more potent, although less specific. Efforts have been made to construct more potent and specific immunotoxins by separating the toxin B-chain domains involved in cell binding from the domains involved in A-chain entry.
Target cell toxicity of immunotoxins can be increased by including the toxin B-chain in the antibody-toxin complex or by adding it separately. To achieve maximal in vitro target-cell selectivity with immunotoxins containing intact ricin, lactose must be added to the medium to block non-target-cell binding and toxicity of the immunotoxin via the ricin B-chain. This approach is feasible in those clinical settings, such as bone marrow transplantation, where the target cell population can be incubated in vitro in the presence of lactose. Without blockage of the B-chain binding domain, however, whole toxin conjugates have a high degree of non-target-cell toxicity, thereby limiting their usefulness in vivo.
Construction of reagents that combine the potency of intact toxin conjugates with the cell-type selectivity of toxin A-chain conjugates may be possible if the binding site on the toxin B-chain could be irreversibly blocked. Covalent and noncovalent chemical modifications that block the binding activity of ricin intracellularly also block its entry function, suggesting that the binding and translocation functions may be inseparable.
Previously, domain deletion was unsuccessfully used in an attempt to separate the translocation and the binding functions of diphtheria toxin B-chain. Immunotoxins made with the A-chain, intact diphtheria toxin, and a cloned fragment of diphtheria toxin (MspSA) that lacks the C-terminal 17-kDa region of the B subunit were compared. The intact diphtheria conjugate was 100 times more toxic than the MspSA conjugate was, which, in turn, was 100-fold more toxic than was the diphtheria toxin A-chain conjugate. The C-terminal, 17-kDa region, which contains the cell surface binding site, therefore potentiates immunotoxin activity 100-fold. It has not been possible to determine whether this C-terminal translocation activity was distinct from the binding activity.
Laird and Groman, J. Virol. 19: 220 (1976) mutagenized Corynebacterium with nitrosoguanidine and ultraviolet radiation and isolated several classes of mutants within the diphtheria toxin structural gene. Leppla and Laird further characterized several of the mutant proteins and found that three of them, CRM 102, CRM 103, and CRM 107, retained full enzymatic activity but had defective receptor binding.
Recombinant DNA technology has been used to improve immunotoxin efficacy at the gene level. Greenfield et al. (1984) in Proc. Natl. Acad. Sci. USA 80: 6953-6857, reported that they have cloned portions of diphtheria toxin and created a modified toxin which contains the N-terminal hydrophobic region of diphtheria toxin but lacks the C-terminal cysteine for ease of linking to antibodies. This fragment lacks the cell surface-binding sits of diphtheria toxin but includes most of the hydrophobic region thought to facilitate membrane transport.
Although cleavage of ricin or diphtheria toxin into A and B-chains had been thought to improve the specificity of the immunotoxins produced from the A-chain, cleavage of ricin or diphtheria toxins into A and B-chains removes the portion of the molecule containing residues important for transport into the cytosol of the cell. Specific cytotoxic reagents made by coupling toxin A subunits to antibodies have low systemic toxicity but also very low tumor toxicity. More potent reagents can be made by coupling intact toxins to monoclonal antibodies, as detailed in J. Immunol. 136: 93-98 and Proc. Natl. Acad. Sci. USA 77: 5483-5486. These reagents, however, have a high systemic toxicity due to the toxin binding to normal cells, although they can have applications in vitro in bone marrow transplantation (cf. Science 222: 512-515).
It was found by Youle et al., as reported in Jour. Immunol., op. cit., that monoclonal antibody-intact diphtheria cell conjugates reacted quite differently from the intact ricin immunotoxins. Of the four reagents examined, a monoclonal antibody against type T3 antigen linked to diphtheria toxin (UCHT1-DT) had unique properties. This reagent showed greater selectivity in its toxicity to T cells as compared to stem cells than UCHT1-ricin. UCHT1-DT was found to be 10 to 100 times more selective than any previously reported immunotoxin.
Neville et al., in U.S. Pat. Nos. 4,359,457 and 4,440,747, disclose that the receptor specificity of toxins can be altered by coupling the intact toxin to monoclonal antibodies directed to the cell surface antigen Thy 1.2. However, the only toxin specifically disclosed to be treated in this manner is ricin. The same inventors in U.S. Pat. No. 4,500,637, disclose the covalent linkage of a monoclonal antibody known as TA-1 directed against human T-cells for use in treating human donor bone marrow before the marrow is fused into a human recipient. Thus, this reagent has been found to be useful in preventing graft versus host disease.
Another method of treating ricin to increase the rate of protein synthesis inhibition is by adding excess ricin B-chain to target cells independent of the amount of ricin A-chain bound to the cell surface membrane. The ricin A-chains used in this procedure are conjugated to anti-Thy 1.1 monoclonal antibodies. This process is disclosed in Neville et al., U.S. Pat. No. 4,520,011.
Yet another method of treating graft versus host disease is disclosed in Neville et al., U.S. Pat. No. 4,520,226. In this method, monoclonal antibodies specific for T-lymphocytes in human donor bone marrow are covalently linked to separate ricin toxin, combined in a mixture to form a treatment reagent, and combined with bone marrow removed from a human donor. The bone marrow-reagent mixture is then infused into an irradiated recipient, which virtually eliminates T-lymphocyte activity.
However, none of the prior art has shown effective immunotoxins prepared from diphtheria toxin which have the desired specificity and activity.